Methods of producing t cell populations enriched for stable regulatory t-cells

ABSTRACT

The present invention provides methods for producing cell populations enriched for stable, regulatory T cells (Tregs). In particular, the invention relates to methods for culturing T cells such that the final culture is enriched for stable, regulatory T cells. It also relates to methods for stabilizing regulatory T cells. Also provided are compositions enriched for stable, regulatory T cells, which are useful for treating individuals in need of such treatment. The methods and compositions disclosed herein can also be used to treat an individual suffering from an immune-mediated disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a non-provisional patent application which claimsthe benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication Ser. No. 61/576,837, filed Dec. 16, 2011, entitled “METHODSOF PRODUCING T CELL POPULATIONS ENRICHED FOR STABLE REGULATORY T-CELLS,”the disclosure of which is hereby incorporated herein by reference inits entirety.

FIELD OF INVENTION

The present invention relates to methods of producing compositions thatare enriched for regulatory T-cells, and the use of such compositions intreating disease.

BACKGROUND

Foxp3⁺ Tregs are a unique subset of CD4⁺ T cells responsible forself-tolerance and for the prevention of autoimmune disease (Shevach EM, Immunity, 2009; 30(5):636-645). Adoptive Treg infusion has beensuggested as a potential therapy for the prevention of Graft versus HostDisease (GVHD) following stem cell transplantation, organ allograftrejection, and for the treatment of autoimmune diseases such as type Idiabetes and multiple sclerosis (Roncarolo M-G, Battaglia M., Nat RevImmuno., 2007; 7(8):585-598; Riley J L, June C H, Blazar B R, Immunity,2009; 30(5):656-665). Adoptive transfer of Foxp3⁺ Tregs in mouse modelshas been shown to prevent acute and chronic GVHD without negativeeffects on the graft versus leukemia response (Hoffmann P, Ermann J,Edinger M, Fathman C G, Strober S, J Exp Med, 2002; 196(3):389-399).More recently, a number of groups have reported that co-transfer ofexpanded Tregs from umbilical cord samples (Brunstein C G, Miller J S,Cao Q, et al., Blood, 2011; 117(3):1061-1070) or from peripheral bloodappears to be both safe (Trzonkowski P, Bieniaszewska M, Jukinska J, etal., Clin Immunol, 2009; 133(1):22-26) and in one study remarkablyeffective in preventing acute GVHD following stem cell transplantation(Di Ianni M, Falzetti F, Carotti A, et al., Blood, 2011;117(14):3921-3928).

Although considerable enthusiasm has been generated for adoptive Tregtherapy, several major issues remain to be resolved. First, mostclinical applications of Treg therapy will require large numbers ofcells and optimal methods for Treg expansion are now being explored.Expansion of highly purified populations of human Tregs also frequentlyresults in loss of Foxp3 expression during the expansion process.Secondly, in contrast to studies in the mouse, Foxp3 expression can bereadily induced during in vitro stimulation of conventional human Tcells (Shevach E M, Tran D Q, Davidson T S, Andersson J, Eur J Immunol,2008; 38(4):915-917). T cells induced in vitro to express Foxp3frequently lack a Treg phenotype, continue to make effector cytokinesand lack in vitro suppressive function (Shevach E M, Tran D Q, DavidsonT S, Andersson J, Eur J Immunol, 2008; 38(4):915-917). Thus, expressionof Foxp3 cannot be considered a completely reliable marker forfunctional human Tregs.

A number of approaches have been used to address these problems.Combined use of several surface markers (CD127^(lo) and CD25^(hi)) hasfacilitated isolation of more highly enriched populations of Foxp3⁺ Tcells with less contamination by CD25^(int) activated T cells (Liu W,Putnam A L, Xu-Yu Z, et al., J Exp Med. 2006; 203(7):1701-1711).Addition of inhibitors of the mTOR pathway, such as rapamycin, block theexpansion of contaminating conventional T cells and favor the expansionof Tregs, but purity greater than 60% is rarely achieved after severalrounds of expansion depending on the starting population (Hippen K L,Merkel S C, Schirm D K, et al., American Journal of Transplantation,2011; 11(6): 1148-1157). CD4⁺CD25⁺CD45RA⁺Foxp3⁺ T cells, although aminor subpopulation (5-30%) of the Foxp3⁺ pool in adults, appear to havea greater propensity to expand in culture and have enhanced stability ofFoxp3 expression compared to CD4⁺CD25⁺CD45RO⁺Foxp3⁺ T cells (Miyara M,Yoshioka Y, Kitoh A, et al., Immunity, 2009; 30(6):899-911).

Foxp3⁺ Tregs can be divided into two potentially distinctsubpopulations. One population is generated in the thymus and has beentermed natural (n)Tregs. A second population is generatedextrathymically in peripheral sites and has been termed induced (i)Tregs or adaptive Treg. It has recently (Thornton A M, Korty P E, Tran DQ, et al., J. Immunol. 2010; 184(7):3433-3441) been demonstrated thatthe transcription factor, Helios, a member of the Ikaros genesuperfamily, is expressed in 70% of both mouse and human Foxp3⁺ T cells.Foxp3⁺ Helios⁻ T cells are primarily iTregs as Foxp3⁺ T cells induced invitro are Helios⁻, and Foxp3⁺ T cells induced in vivo in response tooral antigen administration, antigen administered i.v., or T cellsactivated in response to lymphopenia are almost exclusively Helios⁻.

Currently, there is no reliable method for producing populations offunctional, human Tregs that can be used for treating disease. Thepresent invention provides such a method.

SUMMARY OF INVENTION

The present invention relates to a method for producing a population ofcells enriched for stable, regulatory T cells. In one embodiment, themethod comprises culturing isolated cells comprising an initialpopulation of regulatory T-cells, in the presence of an oligonucleotide,to expand the initial regulatory T-cells. In one embodiment, theoligonucleotide is an oligodeoxynucleotide. In one embodiment, theoligonucleotide is between 11 and 49 nucleotides in length. In oneembodiment, the oligonucleotide is between 15 and 40 nucleotides inlength. In one embodiment, the oligonucleotide is between 20 and 30nucleotides in length. In a preferred embodiment, the oligonucleotide is25 nucleotides in length.

In one embodiment, the method is conducted using isolated peripheralblood mononuclear cells. In a further embodiment, the isolated cells arelymphocytes. In a further embodiment, at least some of the isolatedcells are CD4+Foxp3+. In yet a further embodiment, the isolated cellsare also Helios+. In one embodiment, at least some of the isolated cellshave a characteristic selected from the group consisting of being CD4positive, being CD25^(hi), and being CD127⁻. In a further embodiment, atleast some of the isolated cells have a characteristic selected from thegroup consisting of being Foxp3 positive and being Helios positive.

In one embodiment, expansion of the initial regulatory T-cells resultsin a population of cells in which at least 60% of the cells are stable,regulatory T-cells. In one embodiment, expansion of the initialregulatory T-cells results in a population of cells in which at least70% of the cells are stable, regulatory T-cells. In one embodiment,expansion of the initial regulatory T-cells results in a population ofcells in which at least 80% of the cells are stable, regulatory T-cells.In one embodiment, expansion of the initial regulatory T-cells resultsin a population of cells in which at least 90% of the cells are stable,regulatory T-cells. In one embodiment, expansion of the initialregulatory T-cells results in a population of cells in which at least95% of the cells are stable, regulatory T-cells.

Another embodiment of the present invention is a method for stabilizingT regulatory cells. In one embodiment, the method comprises culturingisolated cells comprising an initial population of regulatory T-cells,in the presence of an oligonucleotide to expand the initial regulatoryT-cells.

Another embodiment of the present invention is a composition comprisingisolated cells, wherein at least 60% of the isolated cells are stable,regulatory T-cells. In one embodiment, composition is produced byculturing isolated cells comprising an initial population of regulatoryT-cells, in the presence of an oligonucleotide to expand the initialregulatory T-cells. In one embodiment, the stable, regulatory T-cellsare Foxp3+ Helios+.

Another embodiment of the present invention is a method for treating anindividual for an autoimmune disease, the method comprisingadministering a composition comprising isolated cells, wherein at least60% of the isolated cells are stable, regulatory T-cells. In oneembodiment, composition is produced by culturing isolated cellscomprising an initial population of regulatory T-cells, in the presenceof an oligonucleotide to expand the initial regulatory T-cells. In oneembodiment, the stable, regulatory T-cells are Foxp3+ Helios+.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Foxp3⁺Helios⁺ Tregs have Uniformly Demethylated TSDR and do notProduce Inflammatory Cytokines.

(A) Cells were isolated from the buffy coat of a male donor,fixed-permeabilized, and stained for intracellular expression of Foxp3and Helios. Stained cells were sorted into the 4 different fractions.Upper plot shows the staining and gating condition before sorting. ForDNA methylation analysis of the TSDR, bisulfite modification of genomicDNA was performed after extraction from the sorted fractions.Methylation of CpG was read and analyzed by the pyrosequencing method.Results from 7 donors are shown in the lower plot. (B) CD4⁺CD25⁺ cellswere sorted from the buffy coat and were stimulated with plate-boundanti-CD3 and anti-CD28 antibodies for 5 days. The cells were theexpanded for an additional 5 days with anti-CD3/anti-CD28antibody-coated magnetic beads (CD3/CD28-beads). Intracellular stainingfor cytokine production was performed after restimulation with PMA andionomycin. Data shown is one of three independent experiments.

FIG. 2. Addition of CpG ODN Increases the Frequency of Foxp3⁺Helios⁺Cells During In Vitro Expansion.

(A) FACS-sorted CD4⁺CD25⁺ cells (square gate in left plot) werestimulated with plate-bound anti-CD3 and anti-CD28 in the presence ofTGFβ (5 ng/ml) and CpG ODN (ODN2395, 2.5 μM) for 5 days. The cells werethen washed and incubated for two additional days in IL-2 containingmedia. Foxp3 and Helios staining was performed on gated viable CD4⁺cells. (B) Flow cytometry data showing that CpG ODN, not TGFβ, mediatesthe enhancement of the frequency of Foxp3⁺Helios⁺ cells. (C) Flowcytometry data showing that the CpG ODN induced frequency ofFoxp3⁺Helios⁺ cells is maintained during prolong expansion cultures.Cells were sorted as in (A), stimulated for 5 days with plate-boundanti-CD3/CD28, washed, and then expanded for the indicated periods oftime with anti-CD3/CD28 beads and IL-2. (D) Graph showing that CpG ODNenhances the frequency of Foxp3⁺Helios⁺ cells in both freshly isolatedand pre-activated T cells. Cells were isolated and stimulated for 5 daysas in (A). The CpG ODN was added at the time points indicated in thefigure.

FIG. 3. The Foxp3⁺Helios⁺ T Cells Expanded in the Presence of the CpGODN are Functional Tregs.

(A) The TSDR of CD4⁺CD25⁺ T cells expanded in the presence of the CpGODN is fully demethylated. Same protocol as in FIG. 1 a except thatcells are expanded for 9 days. (B) The 9 day expanded cells were treatedas in FIG. 1 b with PMA/ionomycin and analyzed for intracellularcytokines expression. (C) The population of cells expanded in thepresence of the CpG ODN exhibits greater suppressive activity than cellsexpanded in the absence of the ODN. Tregs were expanded as in (A) andcultured for three days at different ratios with freshly isolatedCD4⁺CD25⁻ cells (5×10⁴ cells/well) in the presence of γ-irradiated PBMCs(5×10⁴) and soluble anti-CD3 (2 μg/ml). ³H-TdR incorporation wasmeasured during the last 18 hours of culture.

FIG. 4. An Increased Frequency of Foxp3⁺Helios⁺ T Cells is Seen in thePresence of the CpG ODN During Expansion of Distinct Populations ofTregs.

(A) Sorting strategy used for isolation of CD127^(hi)CD25⁺,CD127^(lo)CD25^(int), and CD127^(lo)25^(hi) T cells. Expression of Foxp3and Helios was analyzed in the different populations after sorting. (B)CpG ODN-mediated enhancement of the frequency of Foxp3⁺Helios⁺ cells inthe sorted populations. (C) Sorting strategy for the isolation ofCD25⁺CD45RA⁺ and CD25⁺CD45RA⁻ Treg subpopulations. Expression of Foxp3and Helios in the cells was performed after sorting. (D) CpGODN-mediated enhancement of the frequency of Foxp3⁺Helios⁺ cells in thesorted CD25⁺CD45RA⁺ and CD25⁺CD45RA⁻ Treg subpopulations.

FIG. 5. CpG ODN Acts Directly on Foxp⁺Helios⁺Cells.

The enhancement of the frequency of Foxp3⁺Helios⁺ Tregs by CpG ODN isnot secondary to differential effects of the ODN on the proliferation ofFoxp3⁺Helios⁺, Foxp3⁺Helios⁻, or Foxp3⁻Helios⁻ subpopulations. A mixturecontaining 50% CD4⁺CD25⁺ and 50% CD4⁺CD25⁻ T cells was created aftersorting. Following labeling with CFSE, the mixture was stimulated withplate-bound anti-CD3/CD28 in the presence or absence of the CpG ODN (2.5μM). The extent of CFSE dilution was analyzed by gating on the threesubsets (Foxp3⁺Helios⁺, Foxp3⁺Helios⁻, and Foxp3⁻Helios⁻).

FIG. 6. Random ODNs Also Stabilize Foxp3⁺Helios⁺ T Cells.

(A) Both a TLR9 agonist and a TLR9 antagonist stabilize Helios⁺ Foxp3⁺Tregs. Cells were isolated as in FIG. 5 a and stimulated cells in thepresence of 2.5 μM CpG ODN or ODN TTAGGG for the indicated time. (B)TLR7 and TLR8 agonists do not stabilize Helios⁺Foxp3⁺ Tregs. Cells wereisolated as in panel a and stimulated in the presence of CpG ODN, or 10μM of ssDNA40 (CL264, TLR8 agonist) or ssDNA40 (TLR7 agonist) or ssRNA41(negative control for ssDNA40). (C) Random sequence ODNs stabilizeHelios Foxp3⁺ Tregs. Left panel, CD4⁺CD127^(lo)CD25⁺ cells were sortedand stimulated for 5 days in the presence of 2.5 μM of ODN TTAGGG or ODNNNNGGG. The cells were then expanded with anti-CD3/CD28 beads in theabsence of the ODN and stained for Foxp3 and Helios expression 8 dayslater. Right panel, same conditions as in left panel except thatdifferent ODNs were added (4× indicates 4-times repeat (24 mers) of 6 bynucleotides). (D) Stabilization of Foxp3 and Helios expression by ODNrequires physical stability of the ODN, but no specific basecomposition. CD4⁺CD127^(lo)CD25^(hi) cells were stimulated and expandedas same as panel (C) in the presence of phosphorothioate backboned ODN(ODNps), phosphodiester backboned ODN (ODNp), biotin-conjugated ODN at5′ end (Biotin-5-ODN), or ODNs w/o N (G, A, T, or C), indicatingrandomly synthesized ODN with exclusion of N nucleotide (G, A, T, or C).All ODNs used are 24mers at a concentration of 2.5 μM. (E) ˜25 base-pairODNs are optimal for stabilization of Foxp3 and Helios expression.CD4⁺CD127^(lo)CD25⁺ cells were isolated as in panel (A), stimulated for5 days in the presence of different length ODNs (10mer, 10-bp long ODN;25mer, 25-bp long ODN; 50mer, 50-bp long ODN; 100mer, 100-bp long ODN).Simulated cells were expanded for an additional 12 days in the absenceof the ODN were treated during 5 day of stimulation, then, stimulatedcells were expanded for 7 more days in the absence of ODNs, and stainedfor Foxp3 and Helios expression. Data shown is the one of 4 independentexperiments. (F) ODNs are localized in cytosol of Tregs. MACS-sortedCD4⁺ T cells were isolated from buffy coat, stimulated with plate-boundanti-CD3/CD28 for 18 hours in the presence of Biotin-5-ODN or unlabeledODN and then washed 3 times in FACS staining buffer. Extra- andintracellular Biotin-5-ODN in stimulated cells was stained withdifferent fluorescence-conjugated strepavidins. For the microscopicanalysis, fixed and stained cells were mounted with DAPI-containingmounting solution (scale 200×).

FIG. 7. Quantitative RT-PCR Analysis of Endosomal TLR's.

Conventional T cells (Tconv, CD4⁺CD25⁻CD127⁺) and Treg (CD4⁺CD25⁺CD12T⁻)were isolated from buffy coats by FACS sorting. The purity of theisolated populations was verified by analysis of Foxp3 and Heliosexpression (Top plots). Extraction of total RNA was done using TRIZOL.Expression of TLR7, TLR8, and TLR9 were determined by quantitativeRT-PCR. For normalization of variation between sample tubes, 18s rRNAwas amplified as internal gene control.

FIG. 8. Methylation Status of the TSDR in Expanded Treg Subpopulations.

CD4⁺CD25⁺ Tregs were isolated from a male donor and were expanded withanti-CD3/CD28 for 9 days. The cells were then fixed and stained withanti-Foxp3 and anti-Helios. The 4 different subsets (Foxp3⁺Helios⁺,Foxp3⁺Helios⁻, Foxp3⁻Helios⁺, Foxp3Helios⁻) were sorted (left plot).Extraction of genomic DNA and reading the methylation status of the TSDRwas performed as described in Methods. Each bar indicates percentmethylation (mean±SD) of the 11 CpGs analyzed.

FIG. 9. Effect of Initial CpG ODN Treatment on the SubsequentProliferative Responses of Treg Subpopulations in the Absence of ODN.

Tregs (CD4⁺CD127^(lo)CD25⁺CD45RA⁺) were isolated and stimulated for 5days in the presence or absence of CpG ODN (2.5 μM). Stimulated cellswere washed twice in complete media, and labeled with CFSE. CFSE-labeledcells (Foxp3⁺Helios⁺, Foxp3⁺Helios⁻, and Foxp3⁻Helios⁻) were thenstimulated with anti-CD3/CD28 beads for 3 more days in the absence ofODNs and their proliferative responses measured by CFSE dilution. Lefttop plots shows Foxp3 and Helios expression after the initial 5day-expansion. Right top plots show Foxp3 and Helios expression afterthe second stimulation in the absence of CpG ODN. Left bottom histogramindicates CFSE labeling of each population before second culture. Rightthree bottom histograms show the CFSE dilution plots of eachsubpopulation after the second 3-day culture in the absence of the ODN.

FIG. 10. Effect of ODNs of Different Length on Stabilization of Foxp3Expression.

Experiment was performed as described in FIG. 6 e. Top graph indicatesthe total cells recovered (mean±SD) from cultures in the presence of theODN. Bottom graph indicates the percent (mean±SD) of viable cells in thesame cultures. Similar results were obtained with cells isolated from 3different donors.

FIG. 11. ODN Cannot be Detected in Foxp3+Helios+ Cells after 72 Hours ofExpansion in the Absence of ODN.

CD4⁺CD127⁻CD25^(hi) cells were isolated and stimulated for days in thepresence of ODN (2.5 μM). Stimulated cells were washed in fresh culturemedia twice and resuspended in IL-2 containing media and expanded in theabsence of ODN. At the indicated time, expanded cells were harvested,fixed, permeabilized, and stained with Biotin-conjugated ODN andFITC-Streptavidin. The result is one of three experiments with cellsfrom different donors.

FIG. 12. Stabilization effect of ODN is consistently observed acrossindividuals regardless and is independent of age. FACS-sorted CD4⁺CD25⁺cells (square gate in left plot) were stimulated with plate-boundanti-CD3 and anti-CD28 in the presence of ODN (ODN2395 orcustom-synthesized ODN, 2.5 μM) for 5 days. The cells were then washedand incubated for two additional 7 days in IL-2 containing media. Foxp3and Helios staining was performed on gated viable CD4⁺ cells. Donors(n=15) ranged in age from 19-75 and were all male.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel method for producing T cellsthat are useful for treating diseases related to the immune system. Morespecifically, the present invention relates to the inventor's discoverythat the presence of oligodeoxynucleotides (ODNs) having particularproperties, during expansion of certain lymphocyte populations in cellculture, results in an expanded cell population that is enriched forregulatory T cells (Tregs). Such populations of cells are useful forpreventing or treating disease such as Graft versus host Disease (GVHD)and autoimmune diseases such as, for example, type I diabetes andmultiple sclerosis.

Before the present invention is further described, it is to beunderstood that this invention is not strictly limited to particularembodiments described, as such may of course vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It should further be understood thatas used herein, the term “a” entity or “an” entity refers to one or moreof that entity. For example, a nucleic acid molecule refers to one ormore nucleic acid molecules. As such, the terms “a”, “an”, “one or more”and “at least one” can be used interchangeably. Similarly the terms“comprising”, “including” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

Methods of the present invention can generally be practiced by culturingisolated cells that comprise a population of T-cells, in the presence ofan oligonucleotide having particular characteristics, to expand at leasta portion of the T-cell population. In preferred embodiments, theisolated cells comprise an initial population of regulatory T-cells, andthe culture conditions result in expansion of at least a portion of theinitial regulatory T-cells. The result of such methods is an expandedpopulation of cells that is enriched for stable, regulatory T-cells.These enriched cells can be used to treat disease related to the immunesystem.

Accordingly, one embodiment of the present invention is a method toproduce a population of cells having stable, regulatory T-cells (Tregs),the method comprising isolating cells comprising an initial populationof regulatory T-cells, and culturing the isolated cells in the presenceof an oligonucleotide having particular characteristics, to expand atleast some of the initial, regulatory T-cells.

As used herein, the terms isolated, isolating, purified, and the like,do not necessarily refer to the degree of purity of a cell or moleculeof the present invention. Such terms instead refer to cells or moleculesthat have been separated from their natural milieu or from components ofthe environment in which they are produced. For example, a naturallyoccurring cell or molecule (e.g., a T cell, a DNA molecule, etc.)present in a living animal, including humans, is not isolated. However,the same cell, or molecule, separated from some or all of the coexistingmaterials in the animal, is considered isolated. As a further example,according to the present invention, cells that are present in a sampleof blood obtained from a person would be considered isolated. It shouldbe appreciated that cells obtained from such a sample using furtherpurification steps would also be referred to as isolated, in keepingwith the notion that isolated does not refer to the degree of purity ofthe cells.

With further regard to the present invention, isolated cells useful forpracticing the disclosed methods can be any isolated cells that compriseT-cells, and in particular, regulatory T-cells. Such cells can beobtained as a sample from an animal, including humans, or they can beobtained from cells in culture. Examples of cell samples useful forpracticing the present invention include, but are not limited to, bloodsamples, lymph samples, and tissue samples. In one embodiment, theisolated cells are obtained from a blood sample. In another embodiment,the isolated cells are obtained from cells in culture.

It is known in the art that T-cells belong to the class of cells knownas lymphocytes, which are a type of agranulocyte. Agranulocytes, alsoknown as mononuclear leukocytes, are characterized by the absence ofgranules in their cytoplasm. The lymphocytes comprise at least threeseparate cell types: B-cells, T-cells and natural killer cells.Furthermore, T-cells can be further divided into effector T cells andregulatory T-cells. In various embodiments, the isolated cells cancomprise mononuclear, agranulocyte or lymphocyte cell populations, solong as they comprise T-cells, and in particular, regulatory T-cells(also referred to as Tregs). As used herein, Tregs are a subpopulationof T-cells that suppress activation of the immune system and express, atleast, the transcription factor Foxp3. Tregs suppresses cytokineproduction and proliferation of T effector cells. Tregs do not expressinflammatory cytokines such as interferon-γ, interleukin-17, andinterleukin-2 and do not proliferate when stimulated via the T cellreceptor in vitro.

Several methods are used to identify Tregs. For example, all Tregsexpress the CD4 and CD25 proteins, and thus are CD4+ and CD25+. Suchproteins are therefore referred to as markers, or marker proteins, forTregs. Thus, in one embodiment, the isolated cells comprise Tregs thatare at least CD4+CD25+. Such cells make up about 5-10% of the matureCD4+ T-cell population in humans, and about 1-2% of CD4+ cells in wholeblood. However, because the CD25 protein can also be expressed onnon-regulatory cells during activation of the immune system, a moreaccurate identification of Tregs in a cell population can be made bydetecting expression of the transcription factor protein, forkhead boxp3 (Foxp3). Thus, in one embodiment, the isolated cells comprise Tregsthat are at least CD4+CD25+Foxp3+. A small percentage of Tregs mayexpress Foxp3, but express low to undetectable levels of CD25. Detectionof the presence or absence of other marker proteins can improve thisanalysis even further. Such markers include, for example, Helios (amember of the Ikaros family of zinc finger proteins) and CD127. Withregard to CD127, the absence or low (10) levels of expression of thisprotein, as compared to intermediate (int) or high (hi) levels ofexpression, indicates the T-cell is a Treg. Methods of determiningwhether the expression level of CD127 is low, intermediate, or high, aredisclosed herein and are known to those skilled in the art. For exampleLiu W, Putnam A L, Xu-Yu Z, et al. CD127 expression inversely correlateswith FoxP3 and suppressive function of human CD4+ T reg cells. J ExpMed. 2006; 203(7):1701-1711, which is incorporate herein by reference,teaches that low level-expression of CD127 is one of phenotypic featureof peripheral blood-resident Tregs in healthy donors and patients andallows one to distinguish Foxp3+ Treg from Foxp3− effector cells.Accordingly, this references teaches assays for measuring the level ofexpression of CD127. In various embodiments, the isolated cells compriseTregs that have at least one characteristic selected from the groupconsisting of: (i) being Helios+, (ii) being CD127−; and, (iii) beingCD127^(lo). In one embodiment, the isolated cells comprise Tregs thatare CD4+CD25+/−Foxp3+Helios+. In a further embodiment, the isolatedcells comprise Tregs that are CD4+CD25+/−Foxp3+Helios+CD127−. In anotherembodiment, the isolated cells comprise Tregs that areCD4+CD25+/−Foxp3+Helios+CD127^(lo). In addition to the markers alreadydescribed, Tregs of the present invention may also express high levelsof cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) andglucocorticoid-induced TNF receptor (GITR).

In addition to detecting the presence, absence of expression level ofvarious protein markers, Treg's can be identified using methylationanalysis. For example, in Treg cells, a region of the foxp3 gene isdemethylated. This region is referred to as the Treg-specificdemethylated region (TSDR). Methods of detecting methylation ordemethylation of this region are disclosed in the art. (see, forexample, Polanski et al., Methylation matters: binding of Ets-1 to thedemethylated Foxp3 gene contributes to the stabilization of Foxp3expression in regulatory Tcells., J. Mol. Med. (2010) 88:1029-1040,which is hereby incorporated by reference). Thus, in one embodiment,isolated cells comprise T-cells in which the TSDR is methylated.

As used herein, the term stable with regard to T-cells refers to T-cellsthat maintain expression of particular markers over several generations.For example, stable Tregs maintain expression of the specific Tregmarkers disclosed herein, over several generations. In one embodiment,stable Tregs are those that maintain expression of CD4 and at least onemarker selected from the group consisting of CD25, Foxp3, Helios,CD127−, and CD127^(lo). In one embodiment, stable Tregs are those thatremain CD4+CD25+Foxp3 over several generations. In one embodiment,stable Tregs are those that remain CD4+CD25+Foxp3 Helios+ over severalgenerations. In one embodiment, stable Tregs are those that remainCD4+CD25+Foxp3 Helios+ and CD127− or CD127^(lo) over severalgenerations.

As used herein, the term generation refers to a round of replication.Thus, a cell that has divided one time has gone through one generation.If the progeny cells then divided once more, the original cells areconsidered to have gone through two generations of replication. The useof such terms is known by those it the art. In one embodiment, stableT-cells are those maintain expression of markers of the presentinvention for at least about 10 generations. In one embodiment, stableT-cells are those maintain expression of markers of the presentinvention for at least about 15 generations. In one embodiment, stableT-cells are those maintain expression of markers of the presentinvention for at least about 20 generations. In one embodiment, stableT-cells are those maintain expression of markers of the presentinvention for at least about 25 generations. In one embodiment, stableT-cells are those maintain expression of markers of the presentinvention for at least about 30 generations. With regard to the numberof generations, the term about is used for convenience and means plus orminus two generations.

The stable expression of markers can also be measured in days. Thus, invarious embodiments, stable T-cells are Treg's that maintain expressionof markers of the present invention for at least about 10 days, at leastabout 15 days, at least about 20 days, at least about 25 days, or atleast about 30 days. With regard to such measurement, the term about isused for convenience and means plus or minus two days.

According to the present invention, once isolated cells are obtained,they are cultured in the presence of an oligonucleotide havingparticular characteristics. It should be noted that isolated cells maybe used directly in the culture step, or they may be further purified orconcentrated prior to being cultured with an oligonucleotide. Forexample, Tregs present in an isolated sample of cells may be identifiedusing molecules, such as antibodies, that bind to Treg markers, therebyallowing the identification of Tregs. The identified Tregs can then beseparated and pooled, or otherwise concentrated, to increase theconcentration of Tregs in the sample. Methods of concentrating cells areknown to those skilled in the art and include, for example, flowcytometry and the use of columns containing molecules that bind Tregmarkers. In one embodiment, the concentration of Tregs is increased byincubating the isolated cells with a molecule that binds Tregs and thenseparating Tregs from non-Tregs by flow cytometry. In such anembodiment, the molecules that bind Treg markers may be labeled with adetectable marker such as, for example, a florescent dye of aradiolabel. Suitable detectable markers are known to those skilled inthe art.

As used herein, the term oligonucleotide refers to a polymer ofnucleotides (or bases). Such oligonucleotides can be synthesized (e.g.,using a nucleic acid synthesizer such as, for example, an AppliedBiosystems Model 380B DNA synthesizer), or it can be generated bydegradation (e.g., chemical or enzymatic digestion, shearing, etc.) of alarger nucleic acid molecule. Preferred oligonucleotides areoligodeoxynucleotides (ODNs). While oligonucleotides of the presentinvention can be any size capable of stimulating enrichment of Tregs ina population of isolated cells, the inventors have found that,surprisingly, oligonucleotides having certain length characteristics,offer advantages over oligonucleotides that are either longer orshorter. For example, the inventors have found that oligonucleotidesthat are too long result in a decrease in the viability of cells exposedto such oligonucleotides. Moreover, oligonucleotides that are too shortdo not stimulate enrichment of Tregs in a population of isolated cells.Thus, in one embodiment, oligonucleotides of the present invention areless than about 300 nucleotides in length, preferably less than about200 nucleotides in length, less than about 100 nucleotides in length,and preferably less than about 50 nucleotides in length. It should benoted that with regard to oligonucleotides of the present invention, theterm about means plus or minus 10%. Further, oligonucleotides of thepresent invention should be at least 10 nucleotides in length. Thus inone embodiment, the oligonucleotide is between 11 and about 199nucleotides in length. In one embodiment, the oligonucleotide is betweenabout 15 and about 99 nucleotides in length. In one embodiment, theoligonucleotide is between about 15 and about 50 nucleotides in length.In one embodiment, the oligonucleotide is between about 20 and about 30nucleotides in length. In various embodiments, the oligonucleotide isselected from the group consisting of an isolated oligonucleotide 21nucleotides in length, an isolated oligonucleotide 22 nucleotides inlength, an isolated oligonucleotide 23 nucleotides in length, anisolated oligonucleotide 24 nucleotides in length, an isolatedoligonucleotide 25 nucleotides in length, an isolated oligonucleotide 26nucleotides in length, an isolated oligonucleotide 27 nucleotides inlength, an isolated oligonucleotide 28 nucleotides in length, and anisolated oligonucleotide 29 nucleotides in length. In a preferredembodiment, the oligonucleotide is 25 nucleotides in length.

Oligonucleotides of the present invention can have any sequence ofnucleotides. That is, the inventors have found that, surprisingly, theability of an oligonucleotide to stimulate enrichment of Tregs in apopulation of isolated cells is independent of its sequence. Thus,oligonucleotides of the present invention may or may not have a pattern.In one embodiment, the oligonucleotide consists of a random sequence ofnucleotides. As used herein, a random nucleotide sequence means that theorder of the nucleotides was not chosen, by a person or machine (e.g.,computer) to have a specific pattern, such as, for example, a proteinencoding sequence, a binding site or a repeating sequence ofnucleotides. That is, at each position in the oligonucleotide, there isan equal probability that any of the four possible nucleotides (i.e.,adenine, guanine, cytosine and thymidine) will be present. As notedabove, while the oligonucleotide can have a random nucleotide sequence,it is not precluded from containing a pattern such as, for example, arepeating run of nucleotides, a protein encoding sequence, aendonuclease recognition site or a binding site. For example, inclusionof a binding motif within, or on the end of, an oligonucleotide may beuseful in purification. Thus, in one embodiment the oligonucleotidecomprises a repeating pattern. In one embodiment, the oligonucleotidecomprises a site selected from the group consisting of a biding motifand a restriction endonuclease recognition site. Similarly, theoligonucleotide is not precluded from being a polymer of a single typeof nucleotide.

It should also be appreciated that oligonucleotides of the presentinvention may be modified to improve, or confer, certain characteristicson the oligonucleotide. For example, modified oligonucleotides may bemore stable or have fluorescent properties. Such modifications can bemade during synthesis of the oligonucleotides or afterwards. Forexample, modified nucleoside triphosphates, such as α-phosphorothioates,2′-O-methyl nucleotides, 7-Deazapurine nucleosides, or 2-aminopurine canbe incorporated into the oligonucleotides during synthesis. Methods ofmodifying nucleic acid molecules are disclosed in Verma and Eckstein,Modified Oligonucleotides Synthesis and Strategy for Users., Annu Rev.Biochem 1998. 67:99-134, which is hereby incorporated by reference.Thus, in one embodiment the oligonucleotide is modified.

Once a suitable oligonucleotide has been obtained, it is cultured withisolated cells of the present invention. According to the presentinvention, culturing (or incubating) the isolated cells in the presenceof the oligonucleotide simply means that the oligonucleotide and thecells are brought together such that they are able to come into contact.Simply as an example of one method of achieving the goals of theinvention, the cells could be placed into a vessel such as an EPPENDORF®tube, along with the oligonucleotide. The mixture could be allowed tosit for a period of time to allow the oligonucleotide and the cells tocome into contact, after which the mixture could be plated or introducedto culture bottles for growth. As an alternative example, the isolatedcells and the oligonucleotide could be introduced directly into cultureplates or bottles. Any such technique can be used, so long as theoligonucleotide and the isolated cells are allowed to come into contact.

Once the oligonucleotide and the cells have been mixed, they are thencultured (or incubated) to allow expansion of at least a portion of theT-cell population present in the isolated cells. Preferably, incubationresults in expansion of at least a portion of the Treg cell populationpresent in the isolated cells. As used herein, expansion of a cellpopulation means that at least one cell within a population is able togrow and divide, resulting in a population of cells retaining thecharacteristics of the original (progenitor) cell(s). Thus, for example,if a culture containing a single cell is expanded for five generations,the expanded culture will contain 32 (2⁵) cells. If the expanded cellsare a stable population of cells, all 32 cells will retain thecharacteristics (e.g., express the same marker proteins, such as, CD4,CD25, Helios, Foxp3, etc) as the progenitor cell. General methods ofculturing cells so that they grow and expand are known to those skilledin the art. Accordingly, it will be appreciated that culture conditionsmay vary depending on the types of cells being expanded, and/or thecharacteristics desired of the expanded cells. With regard to thepresent invention, the cells may be expanded in the presence of certainmolecules that favor, or are necessary for, the expansion of T cells,and in particular Tregs. The requirement of the present method forinclusion of an oligonucleotide has already been described. In oneembodiment, the isolated cells are expanded in the presence of at leastone molecule selected form the group consisting of anti-CDR antibody,anti-CD28-antibody, interleukin-2 (IL-2), inhibitors of the mTORpathway, rapamycin, functional analogs of the afore-mentioned molecules,and mixtures thereof.

Once the appropriate incubation conditions have been established, thecells and oligonucleotide are cultured so that the population of Tregcells expands, yielding a final population of cells that is enriched forregulatory T-cells. As has been previously discussed, only a smallpercentage of mature CD4+ T-cells in humans are Treg's. Moreover, whileapproximately 98% of Treg's present in blood retain theirimmunosuppressive function upon isolation from blood, followingexpansion of such cells using currently available methods, only about15-20% of the expanded cells retain such function. However, the methodsdisclosed herein provide enriched populations of cells in which at least50% or more of the cells are Tregs. As used herein, the term enriched,with respect to T-cell populations, refers to a population of cells inwhich at least about 50% of the cells in the expanded cell populationare stable Tregs. That is, at least 50% of the T-cells in the populationmaintain the ability to suppress immune function. Thus, in oneembodiment, at least about 50% of the T-cells in the population arestable Tregs. In one embodiment, at least about 60% of the T-cells inthe population are regulatory T cells. In one embodiment, at least about70% of the T-cells in the population are stable Tregs. In oneembodiment, at least about 75% of the T-cells in the population arestable Tregs. In one embodiment, at least about 80% of the T-cells inthe population are stable Tregs. In one embodiment, at least about 85%of the T-cells in the population are stable Tregs. In one embodiment, atleast about 90% of the T-cells in the population are stable Tregs. Inone embodiment, at least about 95% of the T-cells in the population arestable Tregs.

As has been described, upon culture of Tregs isolated from blood, alarge percentage of such cells lose markers associated with Tregs.Furthermore, the inventor shave herein described how expansion of suchcells in the presence of an oligonucleotide results in a cultureenriched for stable Tregs. It will be appreciated by those skilled inthe art that loss of Tregs during expansion of isolated cells couldresult from loss of expression of Treg markers, or failure of Tregcells, which by definition express such markers, to expand. Withoutbeing bound by theory, the inventors believe that the oligonucleotideexerts a direct effect on Tregs, thereby stabilizing, or maintaining,expression of Treg markers during the expansion of such cells.Accordingly one embodiment of the present invention is a method tostabilize expression of Treg markers, the method comprising isolatingcells comprising initial regulatory T-cells, and incubating the isolatedcells in the presence of an oligonucleotide of the present invention,under conditions that result in the expansion of at least some of theinitial, regulatory T-cells. Such a method yields progeny Treg cellsthat stably express Treg markers. In one embodiment, the expanded Tregsstably express CD4 and at least one marker selected from the groupconsisting of CD25, Foxp3, Helios, and CD127^(lo). In one embodiment,the expanded Tregs stably express CD4, CD25, and Foxp3. In oneembodiment, the expanded Tregs stably express CD4, CD25, Foxp3 andHelios.

In one embodiment, the expanded Tregs stably express CD4, CD25, Foxp3,Helios, and CD127^(lo).

It has previously been described that the concentration of Tregs in acomposition of isolated cells may be increased prior to incubating theisolated cells with an oligonucleotide. Thus, it should be appreciatedthat concentration of Tregs may also be performed following incubationof the isolated cells with the oligonucleotide. As has been described,concentration of Tregs may be achieved by identifying Tregs using amolecule that binds Treg markers, and then concentrating the identifiedcells using techniques such as, for example, flow cytometry and columnscontaining molecules that bind to Treg markers.

Methods of the present invention result in the production ofcompositions having stable regulatory T-cells, which can be used fortreating various disease related to the immune system. Prior to theinventors discovery, such compositions were impractical, or evenimpossible, to produce, due to various factors such as cost andtechnical difficulties such as the spontaneous instability of functionalTregs (CD4+Foxp3+Helios+). Thus, one embodiment of the present inventionis a composition comprising cells, wherein greater than about 50% of thecells are stable Tregs. One embodiment of the present invention is acomposition comprising isolated T-cells, wherein at least about 60% ofthe T-cells are stable Tregs. In one embodiment, the stable Tregs arepositive for CD4 and at least one marker selected from the groupconsisting of CD25, Foxp3, Helios, CD127−, and/or CD127^(lo). In oneembodiment, stable Tregs are CD4+CD25+Foxp3+. In one embodiment, stableTregs are CD4+CD25+Foxp3 Helios+. In one embodiment, stable Tregs areCD4+CD25+Foxp3 Helios+ and CD127− or CD127^(lo). In one embodiment, thecomposition is produced using a method comprising isolating cellscomprising initial regulatory T-cells, and incubating the isolated cellsin the presence of an oligonucleotide having particular characteristics,under conditions that result in the expansion of at least some of theinitial, regulatory T-cells

Because Tregs are able to suppress activation of the immune system, suchcells can be used to treat an individual having a disease for whichsuppression of the immune system is desirable. Compositions of thepresent invention are particularly useful for treating autoimmunediseases. For example, Tregs can be used to treat or prevent a diseaseor condition such diabetes, multiple sclerosis, graft vs. host disease(GVHD) (e.g., after a bone marrow transplantation), allograft rejectionfollowing tissue transplantation, and the like. Thus, one embodiment ofthe present invention is a method to treat an individual in need of suchtreatment, the method comprising administering a composition comprisingisolated T-cells, wherein at least about 60% of the T-cells are stableTregs. In one embodiment, at least about 70% of the T-cells in thecomposition are stable Tregs. In one embodiment, at least about 80% ofthe T-cells in the composition are stable Tregs. In one embodiment, atleast about 90% of the T-cells in the composition are stable Tregs. Inone embodiment, at least about 95% of the T-cells in the composition arestable Tregs.

The terms individual, subject, and patient are well-recognized in theart, and are herein used interchangeably to refer to a mammal, includingdog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and,most preferably, a human. In some embodiments, the subject has beendiagnosed with an autoimmune disease. In some embodiments, compositionsof the present invention are administered to an individual at risk fordeveloping an autoimmune disease. Such risk can be due to, for example,genetic factors or exposure to environmental factors. Methods ofidentifying individuals at risk for developing an autoimmune disease areknown to those in the art.

The terms individual, subject, and patient by themselves do not denote aparticular age, sex, race, and the like. Thus, individuals of any age,whether male or female, are intended to be covered by the presentdisclosure. Likewise, the methods of the present invention can beapplied to any race, including, for example, Caucasian (white),African-American (black), Native American, Native Hawaiian, Hispanic,Latino, Asian, and European.

Compositions of the present invention are administered using any knownroute used to administer therapeutic compositions, so long as suchadministration results in alleviation of symptoms of an autoimmunedisease. Acceptable protocols by which to administer compositions of thepresent invention in an effective manner can vary according toindividual dose size, number of doses, frequency of dose administration,and mode of administration. Determination of such protocols can beaccomplished by those skilled in the art.

Also included in the present invention are kits useful for practicingthe disclosed methods of the present invention. Thus, one embodiment ofthe present invention is a kit for producing a population of cells thatis enriched for stable, regulatory T-cells (Tregs), the kit comprising(i) oligonucleotides of the present invention and ii) instructions forusing the kit. Kits of the present invention can also comprise variousreagents, such as buffers, necessary to practice the methods of theinvention, as known in the art. Such reagents and buffers may, forexample, be useful for establishing conditions appropriate for expandingisolated cells into enriched populations of Tregs. Thus, such regentsmay include things such as, for example, tissue culture media andimmunoregulatory molecules such as IL-2.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

Example 1

The possibility that instability of Treg cell populations might berelated to the different properties of the Helios⁺ and Helios⁻subpopulations, was explored. One epigenetic marker that has been usedto distinguish nTreg from iTreg is the methylation status of the Tregspecific demethylation region (TSDR) of the Foxp3 gene (Floess S, FreyerJ, Siewert C, et al., PLoS Biol, 2007; 5(2):e38). Demethylation of theTSDR region correlates with the thymic origin of Foxp3⁺ T cells andstable expression of Foxp3, while T cells induced to express Foxp3 invitro have a fully methylated TSDR region and lose expression of Foxp3in vivo or in vitro.

To determine if a correlation exists between Helios expression and TSDRmethylation status, four different CD4⁺ subsets (Foxp3⁺Helios⁺,Foxp3⁺Helios⁻, Foxp3⁻Helios⁺, and Foxp3⁻ Helios⁻) were isolated andexpression of various proteins analyzed. Briefly, human PBMCs wereobtained from 20-80 year-old healthy male donors by Department ofTransfusion Medicine at National Institutes of Health.CD4⁺CD25^(hi)CD127⁻ Tregs were isolated by cell sorting on a FACSAria.Sorted Treg (0.2×10⁶) cells were re-suspended in complete RPMI-1640supplemented with 10% heat-inactivated fetal calf serum, and stimulatedin plates coated with anti-CDR (10 μg/ml) and anti-CD28 (2 μg/ml) in thepresence of IL-2 (150 U/ml) for 5-6 days. Stimulated cells were washedin RPMI 1640-10 media twice and expanded in complete RPMI 1640-10 withrecombinant IL-2 (150 U/ml) for 5-10 days in the presence ofTreg-expander magnetic beads (Cell:Bead=4:1). To evaluate the purity ofsorted Tregs, cells were stained with anti-Foxp3 and anti-Helios(Biolegend) in Foxp3-staining buffer (eBioscience). All flow cytometrywas performed on a FACS LSRII flow cytometer (Becton Dickinson) andanalyzed with FlowJo software (TreeStar).

To determine the methylation status of the TSDR, freshly isolated, or invitro expanded, Tregs were harvested, and washed twice in PBS.Extraction of genomic DNA was performed using the DNeasy Blood & TissueKit (Qiagen). Fixed, stained cells were FACS sorted, washed in PBStwice, and genomic DNA was extracted with QIAamp DNA FFPE tissue Kit(Qiagen). Bisulfite conversion, pyrosequencing, and data analysis weredone by EpigenDx (Worcester, Mass.). 11 CpGs of the human TSDR wereanalyzed (−2376 to −2263 from TSS, ENST00000376207). The percentmethylation of each sample indicates the mean value of all 11 CpGs.

The results of this analysis are shown in FIG. 1A. Foxp3⁻ T cellsexpressed a fully methylated TSDR regardless of Helios expression. Incontrast, Foxp3⁺Helios⁺ cells were fully demethylated in all donors,while the TSDR region of the Fox3⁺Helios⁻ subset was ˜45% methylated.One interpretation of these results is that the Foxp3⁺Helios⁻subpopulation is composed of two subpopulations, one of which expressesa fully methylated TSDR that can potentially lose Foxp3 expression uponin vitro expansion, while the second likely represents a population withgreater stability of Foxp3 expression. These results demonstrate thatCD4+Foxp3+Helios+ Tregs are stable, and possess a demethylated TSDR.

Example 2

Previous work had shown that a low percentage (1-5%) of freshlyexplanted Foxp3⁺ T cells produce effector cytokines (IL-2, IFNγ), andthat almost all the cytokine producing cells were in the Foxp3⁺Helios⁻subpopulation (Liu H, Komai-Koma M, Xu D, Liew F Y, Proc Natl Acad SciUSA, 2006; 103(18):7048-7053). Thus, the ability of cell populationsthat were expanded using the presently disclosed methods was measured.

CD4⁺CD25⁺ T cells were isolated and sorted as described in Example 1,and expanded for 12 days by stimulation with plate-bound anti-CD3 andanti-CD28 in the presence of IL-2. While ˜90% (data not shown) of thestarting population was Foxp3⁺, after 12 days of expansion only 45% ofthe cells remained Foxp3⁺ (FIG. 1B). Upon re-stimulation in vitro withphorbol myristate acetate (PMA)/ionomyin, 20% of the expandedFoxp3⁺Helios⁻ subpopulation produced IL-2 or IFNγ, while the frequencyof cytokine producers remained low in the Foxp3⁺Helios⁺ subpopulation(FIG. 1B). These results suggest that one factor controlling themaintenance of Foxp3 expressing Tregs upon expansion in vitro is theheterogeneity of the starting population. The Foxp3⁺Helios⁻subpopulation appears to contain a high percentage of Tregs withmethylated TSDRs that have the potential for loss of expression ofFoxp3. This same subpopulation also contains a high percentage ofeffector cytokine producing cells that would not be ideal for cellularimmunotherapy.

Example 3

This Example demonstrates that addition of CpG ODN to cell culturesresults in enhanced maintenance of Foxp3+Helios+ cells during in vitroexpansion.

Initially, two potential candidates were selected for testing: TGFβ, andthe toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotide (ODN).CD4⁺CD25⁺ cells, sorted as described in Example 1, were stimulated withplate-bound anti-CD3 and anti-CD28 in the presence of TGFβ (5 ng/ml) orCpG ODN (ODN2395, 2.5 μM) for 5 days. The cells were then washed andincubated for two additional days in IL-2 containing media. Foxp3 andHelios staining was performed on gated viable CD4⁺ cells. The results ofthis analysis, which are shown in FIG. 2, show that the frequency of theFoxp3⁺Helios⁺ positive cells was higher in cells expanded in thepresence of TGFβ and the ODN as compared to cells expanded in theabsence of TGFβ and the ODN (FIG. 2A). The 5 day expanded cells werethen washed, and re-stimulated for an additional 7 days in the absenceof TGFβ and CpG ODN. The results of this analysis, which are shown inFIG. 2B, show that cells initially exposed to TGFβ/ODN maintained ahigher frequency (67.9%) of Foxp3⁺Helios⁺ cells, while cells initiallyexpanded in the absence of TGFβ/ODN had a markedly decreased percentage(34.6%) of the Foxp3⁺Helios⁺ cells. The results further indicate thatCpG ODN was the major factor involved in stabilization of theFoxp3⁺Helios⁺ population, since cells expanded in the presence of TGFβalone had only a modest increase in Foxp3⁺Helios⁺cells, while cellsexpanded with the ODN alone resembled the population treated with thecombination of TGFβ/ODN (FIG. 2B).

To determine the longevity of the effects of the ODN on maintenance ofthe Foxp3⁺Helios⁺cells, FAC-sorted CD4⁺CD25^(hi) cells were expanded inthe presence or absence of the ODN for 5 days, washed, and expanded foran additional 6 or 19 days in the presence of anti-CD3/CD28 beads andIL-2. These results are shown in FIGS. 2C and 2D. Marked enhancement ofthe frequency of the Foxp3⁺Helios⁺cells was observed after 12 days inculture and the enhancement was still prominent after a total of 25 daysin culture even though some decrease in the frequency of theFoxp3⁺Helios⁺cells was observed in the cells initially exposed to theODN (FIG. 2C). It was observed that the continuous presence of the ODNfor the entire 12 days of expansion resulted in a modest increase (75.3%vs 63.7%) in the frequency of Foxp3⁺Helios⁺cells compared to cells thathad just been treated for the initial 5 days (FIG. 2D). Interestingly,when cells that had been previously expanded for 5 days in the absenceof the ODN, were re-stimulated for 7 additional days in the presence ofthe ODN, stabilization (38.1% vs 22.2%) of the frequency ofFoxp3⁺Helios⁺ cells was observed (FIG. 2D). Thus, the ODN exerts astabilizing effect on Foxp3⁺Helios⁺ cells that had been previouslystimulated in the absence of the ODN.

Example 4

This Example demonstrates that Foxp3⁺Helios⁺ cells expanded with CpG ODNare functional Tregs.

Foxp3⁺ Treg cells were expanded in vitro for 25 days and the methylationstatus of the TSDR analyzed, as described in Example 1. The results,which are shown in FIG. 1 and FIG. 8, show that expanded Foxp3⁺Helios⁺cells expressed a fully demethylated TSDR, while the TSDR of theexpanded Foxp3⁺Helios⁻ cells was 70% methylated, a value higher thanthat observed with freshly isolated Foxp3⁺Helios⁻ cells. Next, TSDRmethylation in Foxp3⁺ cells expanded in the presence or absence of theODN for 25 days was compared. The results, which are shown in FIG. 3,show that CpG ODN treatment resulted in a high frequency ofFoxp3⁺Helios⁺ cells (79.7% vs 36%). The TSDR of the entire population ofcells expanded in the presence of the CpG ODN was almost completely(90%) demethylated, while the TSDR of the cells expanded in the absenceof the ODN was 42% methylated.

To determine the effect of long-term expansion on the ability of cellsto produce cytokines, cell populations that had been expanded for 25days in the presence or absence of the ODN were re-stimulated for 4hours with PMA/ionomycin, and cytokine production by gated Foxp3⁺Helios⁺and Foxp3⁺Helios⁻ populations was measured. The results, which are shownin FIGS. 3B and 3 c, show that Foxp3⁺Helios⁺ Tregs expanded in thepresence of the ODN produced low to undetectable levels of IL-2, IFNγ,or IL-17, while Foxp3⁺Helios⁻ cells produced significant levels of allthree cytokines. In addition, the population of Foxp3⁺ T cells that hadbeen expanded in the presence of the ODN, which contained a highpercentage of Foxp3⁺Helios⁺ T cells, exhibited greater suppressiveactivity in a standard in vitro suppression assay (FIG. 3C) than cellsthat had been expanded in the absence of the ODN. Taken together, thesestudies demonstrate that the Foxp3⁺Helios⁺ cells that had been expandedin the presence of the ODN retain all the properties of fully functionalTregs.

Example 5

This example demonstrates the ability of CpG ODN to stabilize Foxp3 andHelios expression in all Treg subsets.

Co-expression of CD25 and low levels of CD127 has been used todistinguish CD25⁺Foxp3⁺ from CD25⁺Foxp3⁻ Teff cells (Hippen K L, MerkelS C, Schirm D K, et al., American Journal of Transplantation, 2011;11(6):1148-1157). In order to determine the relationship betweenFoxp3/Helios expression and the level of expression of CD127, freshlyisolated CD4⁺ T cells were sorted into three different subsets based onexpression of CD25 and CD127, and the relative expression of Foxp3 andHelios analyzed. The results, which are shown in FIG. 4A, show that mostCD127^(hi)CD25^(int) cells did not express either Foxp3 or Helios.However, both CD127^(low) CD25^(hi) and CD127^(low)CD25^(int) containeda high percentage of Foxp3⁺Helios⁺ T cells. To further explore therelationship between these markers, these three subpopulations(CD127^(low) CD25^(hi), CD127^(low) CD25^(int) and CD127^(hi)CD25^(int)T cells) were sorted and expanded in the presence or absence of the CpGODN. The results, which are shown in FIG. 4B, show that ODN had minimaleffects on the composition of the CD127^(hi)CD25″ cells except for aslight increase in the Foxp3⁺Helios⁻ subset (11.3% to 21.7%, left twopanels in FIG. 4B). Addition of the ODN to the expansion ofCD127^(hi)CD25^(int) cells enhanced the recovery of Foxp3⁺Helios⁺ cells(68.2% vs 89%) on day 5 and this enhancement was maintained for anadditional 7 days of culture in the absence of the ODN. The mostinteresting results were observed with the CD127^(low) CD25^(int) cellswhich rapidly lost Foxp3⁺Helios⁺ cells (73% to 22.7%) during the initial5 days of expansion, but retained Foxp3⁺Helios⁺ cells when expanded inthe presence of the ODN (73% to 71%) and maintained this frequency ofFoxp3⁺Helios⁺ cells after a further 7 days expansion in the absence ofthe ODN. These results suggest that the subpopulation of Foxp3⁺Helios⁺cells that express lower levels of CD25 are much less stable than thosethat express higher levels of CD25, but that expansion in the presenceof the ODN exerts a major effect on Foxp3/Helios stabilization in thissubpopulation.

In addition to the use of the levels of CD127 expression to isolateFoxp3⁺ Treg cells, co-expression of CD25 and CD45RA has defined asubpopulation of Foxp3⁺ T cells that are regarded as naïve¹¹, exhibitmore stable Foxp3 expression on expansion in vitro¹⁷, and express fullydemethylated TSDRs (Hoffmann P, Boeld T J, Eder R, et al., Eur JImmunol, 2009; 39(4):1088-1097). Thus, a comparison of Helios expressionin Foxp3⁺ CD45RA⁺ and Foxp3⁺CD45RA⁻ cells revealed that the frequency ofFoxp3⁺Helios⁺ cells was similar in these two subpopulations (data notshown). To further explore this area, CD45RA⁺ and CD45RA⁻ cells weresorted from CD4⁺CD25⁺CD127^(low) peripheral CD4⁺ T cells (FIG. 4C), andall three groups stimulated in the presence or absence of CpG ODN for 6days, washed, and expanded in the absence of the ODN for an additional 6days. The results, which are shown in FIG. 4D, show that after 6 days ofstimulation without ODN, 94% of CD25⁺CD45RA⁺ cells remained Foxp3+,whereas CD25⁺CD45RA⁻ subpopulation began to lose Foxp3 expression (98%to 77.6%). The frequency of Foxp3⁺Helios⁺ cells was also somewhat higherin the CD25⁺CD45RA⁺ subpopulation (81%) than in CD25⁺CD45RA⁻ group(66%). Both cell populations had decreased frequencies of Foxp3⁺Helios⁺T cells after an additional 6 days of expansion in the absence of theODN, although the total frequency of Foxp3⁺ cells remained stable.Addition of the ODN to both groups resulted in enhanced frequencies ofFoxp3⁺Helios⁺ cells during the second 6 days of expansion in the absenceof the ODN. Thus, Foxp3⁺ CD45RA⁺ cells appear to exhibit more stableexpression of Foxp3 upon expansion in vitro, but the frequency ofFoxp3⁺Helios⁺ cells does diminish and can be rescued in both the CD45RA⁺and CD45RA⁻ populations by stimulation in the presence of the ODN.

Example 6

This example demonstrates that CpG ODN directly stabilizeHelios-expressing Foxp3⁺ cells and do not inhibit the proliferation ofFoxp⁺Helios⁻ or Foxp3⁻Helios⁻ cells.

To investigate whether CpG ODN affect cell populations through a directeffect on existing Foxp3⁺ Helios⁺ cells or a suppressive effect on theexpansion of Foxp3⁺ Helios⁻ or Foxp3⁻ Helios⁻ cells that are alsopresent in the starting populations, a starting population containing50% Foxp3⁺ Helios⁺ cells, rather than the higher percentages used in theother studies, was prepared. Such a population of cells allowsobservation of the differential effects of the ODN on Helios⁺ andHelios⁻ cells. The mixture was labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) and the cells stimulated for 6 days in thepresence or absence of the ODN. The results, which are shown in FIG. 5,show that stabilization of the Foxp3⁺Helios⁺ cells was seen afterexpansion in the presence of the ODN (FIG. 5, 30% vs. 11.1%). Additionof the CpG ODN slightly suppressed the proliferation of all threesubsets (Foxp3⁺Helios⁺, Foxp3⁺Helios⁻, and Foxp3⁻Helios⁻) (FIG. 5,histograms on right side). Since there was no significant difference inproliferation between the groups, the results indicate that enhancedfrequency of the Foxp3⁺Helios⁺ subset in the ODN treated group isconsistent with a direct stabilizing effect of the ODN on thissubpopulation.

Example 6

This example demonstrates that ODN stabilizes Helios-expressing Foxp3⁺cells via a cytosomal universal DNA sensor, not via an endosomal TLR.

To determine if the CpG ODN used in our studies mediates its effects onstabilization of Foxp3⁺Helios⁺ T cells by acting via TLR9, which isexpressed in Foxp3⁺ human Tregs (FIG. 7), Tregs were expanded in thepresence of both the TLR9 agonist and a TKR9 antagonist (ODN TTAGGG).Surprisingly, expansion of Tregs in the presence of both the TLR9agonist and a TLR9 antagonist (ODN TTAGGG) resulted in stabilization ofthe Foxp3⁺Helios⁺ subset (FIG. 6A). When both the CpG ODN and the TLR9antagonist were simultaneously added to the expansion cultures, reversalof the stabilization of the Foxp3⁺Helios⁺ subset was not observed (dataare not shown). Although TLR8 or TLR7 expression was not detected inTregs (by RT-PCR), the effects of a TLR8 agonist (ssRNA40) and a TLR7agonist (CL264) were compared with the effect of a TLR9 agonist. Neitherthe TLR8 nor the TLR7 agonists had any effect on the stabilization ofFoxp3⁺Helios⁺ T cells, while the CpG ODN enhanced the frequency of thissubset as noted in our other studies (FIG. 6B). Thus, these resultsindicate that CpG ODN-mediated stabilization of Foxp3⁺Helios⁺ cells ismediated by TLR signaling pathway and that anothernucleotide-recognizing sensor is involved in this stabilization.

To determine the optimal order, or motif, of the ODN sequence needed tostabilize Foxp3⁺Helios⁺ Treg, the TTA sequence from ODN TTAGGG wassubstituted with randomly selected nucleotides (NNN) and the mutantstested for their effect on the stability of Tregs. The results, whichare shown in FIG. 6C, show that ODN NNNGGG behaved in identical fashionto ODN TTAGGG. Moreover, a completely randomized ODN (ODN) alsoefficiently stabilized Foxp3⁺Helios⁺ cells. As the ODN appears to besensed in a non-sequence-specific manner, the role of the nucleotidecomposition on the stabilizing activity of the ODN was examined.Excluding A, T, G, or C from the random synthesized ODN did not diminishthe stabilizing effect (FIG. 6D, bottom panels). In contrast tophosphorothioate ODN (ODNps), phosphodiester ODN (ODNp) failed tostabilize Foxp3⁺Helios⁺ Tregs even when the ODNp was added every 24hours during the 5-day expansion culture (FIG. 6D, top panels, and datanot shown).

Example 7

To determine the optimal size ODN for stabilizing Foxp3⁺Helios⁺ cells,the cell-stabilizing activity of ODNs ranging from 10mer to 100mer wasmeasured. The results, illustrated in FIG. 6E, show that the 10mer hadno activity when compared to the untreated cells, while the 50mer and100mer increased the frequency of the Foxp3⁺Helios⁺ cells after 5 daysof culture, but were also moderately toxic as their use resulted in adecreased cell yield (FIG. 10). Optimal results were obtained in the25mer ODN-treated group including stabilization of the frequency ofFoxp3⁺Helios⁺ cells and an increase in the absolute number of recoveredFoxp3⁺Helios⁺ cells after 12 days of expansion.

Example 8

To determine cellular localization of the ODN sensor, a5′-biotin-conjugated ODN (Biotin-ODN) was synthesized and its locationwithin the cell was determined. As shown in FIG. 6D, the biotin-labeledODN was as active the ODNps in stabilizing expression of Foxp3⁺Helios⁺ Tcells. Purified CD4⁺ T cells were stimulated in the presence ofBiotin-ODN for 18 hours, and then evaluated for intracellular andextracellular expression of the ODN by using two differentfluorescence-conjugated streptavidins (FIG. 6F, left side). In some ofcells, biotin-ODN was detected on the extracellular membrane, but all ofthe cells possessed Biotin-ODN intracellularly. The staining pattern ofBiotin-ODN was identical in Foxp3⁻Helios⁻ and Foxp3⁺Helios⁺ subsets. Onanalysis by confocal microscopy, Biotin-ODN was not detected in thenucleus or associated with the nuclear membrane. All the ODN wasaggregated in granule-like organelles in the cytoplasm (FIG. 6F).Time-course analysis of Biotin-ODN on the FACS indicated that the ODNrapidly disappeared after washing during the expansion phase in theabsence of extracellular ODN (FIG. 11).

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What is claimed:
 1. A method for producing a population of cells havingstable, regulatory T cells, the method comprising culturing isolatedcells comprising an initial population of regulatory T-cells, in thepresence of an oligonucleotide to expand the initial, regulatoryT-cells.
 2. The method of claim 1, wherein the isolated oligonucleotideis between 11 and 49 nucleotides in length.
 3. The method of claim 1,wherein the isolated oligonucleotide is between 15 and 40 nucleotides inlength.
 4. The method of claim 1, wherein the isolated oligonucleotideis between 20 and 30 nucleotides in length.
 5. The method of claim 1,wherein the isolated oligonucleotide is selected from the groupconsisting of an isolated oligonucleotide 21 nucleotides in length, anisolated oligonucleotide 22 nucleotides in length, an isolatedoligonucleotide 23 nucleotides in length, an isolated oligonucleotide 24nucleotides in length, and an isolated oligonucleotide 25 nucleotides inlength.
 6. The method of claim 1, wherein the isolated oligonucleotideis approximately 25 nucleotides length.
 7. The method of claim 1,wherein the isolated cells are peripheral blood mononuclear cells. 8.The method of claim 1, wherein the isolated cells are lymphocytes. 9.The method of claim 1, wherein at least some of the initial regulatory Tcells are CD4+ and Foxp3+.
 10. The method of claim 9, wherein at leastsome of the initial regulatory T cells are also Helios+.
 11. The methodof claim 1, wherein at least some of the initial regulatory T-cells haveat least one characteristic selected from the group consisting of beingCD4 positive, being CD25^(hi), and being CD127⁻.
 12. The method of claim11, wherein at least some of the initial regulatory T-cells have atleast one characteristic selected from the group consisting of beingFoxp3 positive and being Helios positive.
 13. The method of claim 1,further comprising contacting the population of cells with at least onemolecule selected from the group consisting of an anti-CDRε antibody, ananti-CD28 antibody, and interleukin-2.
 14. The method of claim 1,wherein expansion of the initial regulatory T-cells results in anexpanded population of cells in which at least 60% of the cells arestable, regulatory T-cells.
 15. A method for stabilizing T regulatorycells, the method comprising culturing isolated cells comprising initialregulatory T-cells, in the presence of an oligonucleotide to expand theinitial regulatory T-cell population.
 16. A composition comprisingisolated cells, wherein greater than 60% of the isolated cells arestable, regulatory T-cells.
 17. The composition of claim 16, wherein thestable regulatory T-cells are Foxp3+Helios+.
 18. A compositioncomprising a population of stable, regulatory T cells produced accordingto the method of claim
 1. 19. A method of treating an individual for anautoimmune disease comprising administering the composition of claim 16or 18 to the individual.